Triptolide derivatives as immunomodulators and anticancer agents

ABSTRACT

Compounds having the structure I:  
                 
 
are useful for inducing cell death (apoptosis) and in immunosuppression. In structure I, R 1  is H or R, R being selected from lower alkyl, alkenyl, alkynyl, and allenyl, or, R 1  together with R 2 =O (oxo); R 2 =OH, or, R 1  and R 2  together=O (oxo); CR 3 R 5  and CR 4 R 6  are selected from CH 2 , CHOH and CROH; at least one of R 1 , R 5  and R 6  is R; and at least one of CR 3 R 5  and CR 4 R 6  is CH 2 .

This application claims priority to U.S. Provisional Application No.60/434,203, filed Dec. 17, 2002, which is incorporated in its entiretyherein by reference.

FIELD OF THE INVENTION

The present invention relates to compounds useful as immunosuppressive,anti-inflammatory and anticancer agents.

REFERENCES

-   Gleichmann, E. et al., Immunol. Today 5:324 (1984).-   Jung, M. J. et al, U.S. Pat. No. 5,972,998 (1999).-   Jung, M. J. et al, U.S. Pat. No. 6,004,999 (1999).-   Korngold, R. and Sprent, J., J. Exp. Med. 148:1687 (1978).-   Kupchan, S. M. et al., J. Am. Chem. Soc. 94:7194 (1972).-   Kupchan, S. M. et al., U.S. Pat. No. 4,005,108 (1977).-   Lipsky et al., U.S. Pat. No. 5,294,443 (1994).-   Ma et al., J. Chin. Pharm. Sci. 1:12 (1992).-   Murase, N. et al., Transplantation 55:701 (1993).-   Ono and Lindsey, J. Thor. Cardiovasc. Surg. 57(2):225-29 (1969).-   Panchagnula, R. and Thomas, N. S., Intl J of Pharmaceutics 201(2):    131-150 (2000).-   Pu, L. et al., Zhongguo Yaoli Xuebao 11:76 (1990).-   Qi, Y. et al, U.S. Pat. No. 5,663,335 (1997).-   Qi, Y. et al, U.S. Pat. No. 5,962,516 (1999).-   Wang, J. and Morris, R. E., Transplantation Proc. 23:699 (1991).

BACKGROUND OF THE INVENTION

Immunosuppressive agents are widely used in the treatment of autoimmunedisease and in treating or preventing transplantation rejection,including the treatment of graft-versus-host disease (GVHD). Commonimmunosuppressive agents include azathioprine, corticosteroids,cyclophosphamide, methotrexate, 6-mercaptopurine, vincristine, andcyclosporin A. In general, none of these drugs are completely effective,and most are limited by severe toxicity. For example, cyclosporin A, awidely used agent, is significantly toxic to the kidney. In addition,doses needed for effective treatment may increase the patient'ssusceptibility to infection by a variety of opportunistic invaders.

A number of compounds derived from the Chinese medicinal plantTripterygium wilfordii (TW) have been identified as havingimmunosuppressive activity, e.g. in the treatment of autoimmune disease,and in treating or preventing transplantation rejection, including thetreatment of graft-versus-host disease (GVHD), a condition in whichtransplanted marrow cells attack the recipient's cells. See, forexample, co-owned U.S. Pat. No. 6,150,539 (Triptolide prodrugs havinghigh aqueous solubility), U.S. Pat. No. 5,962,516 (Immunosuppressivecompounds and methods), U.S. Pat. No. 5,843,452 (Immunotherapycomposition and method), U.S. Pat. No. 5,759,550 (Method for suppressingxenograft rejection), U.S. Pat. No. 5,663,335 (Immunosuppressivecompounds and methods), and U.S. Pat. No. 5,648,376 (Immunosuppressantditerpene compound), and references cited therein. Such compounds havealso been reported to show anticancer activity. See, for example,Kupchan et al., 1972, 1977, as well as copending and co-owned U.S.application Ser. No. 09/766,156, filed Jan. 19, 2001 and published as USAppn. No. 2002/99051 on Jul. 25, 2002, which is hereby incorporated byreference.

SUMMARY OF THE INVENTION

In one aspect, the invention provides compounds which are useful forimmunosuppressive, anti-inflammatory and anticancer therapy. Thecompounds are derivatives of triptolide represented by Formula I:

where:

R¹ is H or R, where R is selected from lower alkyl, alkenyl, alkynyl,and allenyl, or R¹ together with R²=O (oxo);

R²=OH or together with R¹=O (oxo);

CR³R⁵ and CR⁴R⁶ are selected from CH₂, CHOH and CROH;

at least one of R¹, R⁵ and R⁶ is R; and

at least one of CR³R⁵ and CR⁴R⁶ is CH₂.

In selected embodiments, R is selected from methyl, allyl, and2-propynyl; in one embodiment, R is methyl. In one embodiment, whichincludes C2- or C16-modified triptonides, R¹ together with R²=oxo, oneof CR³R⁵ and CR⁴R⁶ is CROH, and the other is CH₂. In another embodiment,which includes C2- or C16-modified triptolides, R¹ is H, R² is OH, oneof CR³R⁵ and CR⁴R⁶ is CROH, and the other is CH₂.

In a further embodiment, which includes C14-modified triptolides, R¹ isR and R² is OH. Such a compound may also be modified either at C2 or atC16; in one embodiment, neither of these sites is modified; i.e. each ofCR³R⁵ and CR⁴R⁶ is CH₂. When R is methyl, this embodiment includes thecompound designated herein as PG670.

In another aspect, the invention provides a method of effectingimmunosuppression, comprising administering to a subject in need of suchtreatment, in a pharmaceutically acceptable vehicle, an effective amountof a compound having the structure I as described above, including anyof the specific embodiments described above. The invention also providesa method of inducing apoptosis in a cell, by contacting the cell with aneffective amount of a compound having the structure I as describedabove, including any of the specific embodiments described above. Inparticular, the compound may be the compound designated herein as PG670.

These and other objects and features of the invention will become morefully apparent when the following detailed description of the inventionis read in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows dose-dependent induction of apoptosis in Jurkat cells by acompound of the invention (PG670), in comparison with triptolide(Example 2B); and

FIG. 2 shows inhibition of IL-2 production in Jurkat cells by a compoundof the invention (PG670), in comparison with triptolide (Example 3).

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

The terms below have the following meanings unless indicated otherwise.

“Alkyl” refers to a fully saturated acyclic monovalent radicalcontaining carbon and hydrogen, and which may be cyclic, branched orlinear. Examples of alkyl groups are methyl, ethyl, n-butyl, t-butyl,n-heptyl, and isopropyl. Preferably, the alkyl group has one to eightcarbon atoms. “Lower alkyl” refers to an alkyl radical of one to sixcarbon atoms, as exemplified by methyl, ethyl, n-butyl, i-butyl,t-butyl, isoamyl, n-pentyl, and isopentyl, typically one to four carbonatoms.

“Alkenyl” refers to a monovalent or divalent unsaturated, preferablymono-unsaturated, radical containing carbon and hydrogen, and which maybe cyclic, branched or linear. Preferably, the alkenyl group has one toeight carbon atoms. “Lower alkenyl” refers to an alkenyl group havingone to six, typically one to four, carbon atoms.

“Alkynyl” refers to a monovalent or divalent unsaturated, preferablymonounsaturated, radical containing carbon and hydrogen and having atleast one carbon-carbon triple bond. Preferably, the alkynyl group hasone to eight carbon atoms. “Lower alkynyl” refers to an alkynyl grouphaving one to six, typically one to four, carbon atoms.

“Allenyl” refers to a substituent comprising the moiety —CH═C═CH₂.

The term “pharmaceutically acceptable salt” encompasses carboxylatesalts having organic and inorganic cations, such as alkali and alkalineearth metal cations (for example, lithium, sodium, potassium, magnesium,barium and calcium); ammonium; or organic cations, for example,dibenzylammonium, benzylammonium, 2-hydroxyethyl ammonium,bis(2-hydroxyethyl) ammonium, phenylethylbenzylammonium,dibenzylethylene diammonium, and the like. Other cations encompassed bythe above term include the protonated form of procaine, quinine andN-methylglucosamine, and the protonated forms of basic amino acids suchas glycine, ornithine, histidine, phenylglycine, lysine, and arginine.

The term also includes salts formed by standard acid-base reactions withbasic groups, such as amino groups, having a counterion derived from anorganic or inorganic acid. Such counterions include chloride, sulfate,phosphate, acetate, succinate, citrate, lactate, maleate, fumarate,palmitate, cholate, glutamate, glutarate, tartrate, stearate,salicylate, methanesulfonate, benzenesulfonate, sorbate, picrate,benzoate, cinnamate, and the like.

For the purposes of the current disclosure, the following numberingscheme is used for triptolide and triptolide derivatives:

II. Triptolide Derivatives

The compounds of the invention are derivatives of triptolide resultingfrom alkylation at C2, C14, and/or C16, by addition of a carbonnucleophile to a carbonyl group, as described further herein. Thecompounds of the invention may be prepared from triptolide, tripdiolideor 16-hydroxytriptolide. Triptolide can be obtained rom the root xylemof the Chinese medicinal plant Tripterygium wilfordii (TW) or from otherknown sources. The TW plant is found in the Fujian Province and othersouthern provinces of China; TW plant material can generally be obtainedin China or through commercial sources in the United States. Methods forpreparing triptolide, tripdiolide and 16-hydroxytriptolide are known inthe art and are described, for example, in Kupchan et al. (1972, 1977);Lipsky et al. (1994); Pu et al. (1990); and Ma et al. (1992).

The compounds of the invention are represented by Formula I below:

where:

R¹ is H or R, where R is selected from lower alkyl, alkenyl, alkynyl,and allenyl, or R¹ together with R²=O (oxo);

R²=OH or R¹ and R² together=O (oxo);

CR³R⁵ and CR⁴R⁶ are selected from CH₂, CHOH and CROH;

at least one of R¹, R⁵ and R⁶ is R; and

at least one of CR³R⁵ and CR⁴R⁶ is CH₂.

In selected embodiments, R is selected from methyl, allyl, and2-propynyl; in one embodiment, R is methyl. In one embodiment, whichincludes C2- or C16-modified triptonides, R¹ together with R²=oxo, oneof CR³R⁵ and CR⁴R⁶ is CROH, and the other is CH₂. In another embodiment,which includes C2- or C16-modified triptolides, R¹ is H, R² is OH, oneof CR³R⁵ and CR⁴R⁶ is CROH, and the other is CH₂.

In a further embodiment, which includes C14-modified triptolides, R¹ isR and R² is OH. Such a compound may also be modified either at C2 or atC16; in one embodiment, neither of these sites is modified; i.e. each ofCR³R and CR⁴R⁶ is CH₂. When R is methyl, this embodiment includes thecompound designated herein as PG670.

Preferably, when either CR³R⁵ or CR⁴R⁶ is CHOH or CROH, thestereochemistry at C2 or C14, respectively, is such that the hydroxylgroup is depicted above the plane of the page.

The compounds of Formula I can be prepared from the known compoundstriptolide, tripdiolide, and 16-hydroxytriptolide, by oxidation of oneor more hydroxyl groups to keto or aldehyde groups, followed by reactionwith a carbon nucleophile, such as an organolithium or organomagnesiumhalide (Grignard) reagent. Description of oxidizing reagents andprocesses suitable for selective oxidation of alcohols is provided inreferences such as M. Hudlicky, Oxidations in Organic Chemistry (ACSMonograph Series 186, 1990), R. C. Larock, Comprehensive OrganicTransformations (2^(nd) Ed., Wiley, 1999), or J. March, Advanced OrganicChemistry (4^(th) Ed., Wiley, 1992). Strong acidic or basic conditionsshould be avoided. If necessary, the desired product is isolated fromany side products using, for example, HPLC. Several examples are givenbelow.

In Scheme 1, the secondary alcohol at C14 of triptolide is oxidized to aketone (known as triptonide) using, for example, chromiumtrioxide-pyridine complex, CrO₂Cl₂/alumina, or comparable oxidizingreagents. Reaction with methyl lithium (CH₃Li) yields 14-C-methyltriptolide. Preparation and characterization of this compound isdescribed further in Example 1.

In Scheme 1 above, both stereochemistries at C14 are shown, and in thefollowing Schemes, the stereochemistry at the reaction site is notdepicted. However, in reactions in which a chiral center is produced(i.e. additions at C14 and C2), the product resulting from addition“below” the molecule; that is, as in the product on the left in Scheme1, generally predominates. This product is also shown in Example 1below.

In Scheme 2, both secondary alcohols of tripdiolide are oxidized toketones, using an oxidant as described above. Reaction with allyllithium (CH₂═CHCH₂Li) gives both the diol (a), from reaction at bothketones, and the ketol (b), a result of reaction only at the lesshindered ketone.

In Scheme 3, the less hindered alcohol at C2 of tripdiolide is oxidizedto the ketone, using a stoichiometric amount of a reagent such as thosenoted above, under mild conditions. Reaction with allyl lithium givesthe tertiary alcohol at C2.

In Scheme 4, the secondary and primary alcohols of 16-hydroxy triptolideare oxidized with a reagent that will not further oxidize the productaldehyde, e.g. DMSO, pyridinium chlorochromate (Corey reagent), or cericammonium nitrate, to give the keto-aldehyde intermediate. This compoundis reacted with 2-propynyl lithium (CH≡CCH₂Li) to give both the diol (c)and the ketol (d), a result of reaction only at the less hinderedaldehyde.

In Scheme 5, only the primary alcohol of 16-hydroxy triptolide isoxidized, using a selective oxidizing agent such as, for example,RuCl₂(PPh₃)₃; (Me₃SiO)₂/catalytic RuCl₂(PPh₃)₃; DMSO/ClCOCOCl/Et₃N; orDMSO/pyridine SO₃/i-Pr₂NEt (see Larock, cited above). Reaction with2-propynyl lithium gives the diol as shown.

As shown in Scheme 6, below, reaction (of triptonide, in this case) withthe organometallic 2-propynyl reagents can also produce the rearrangedallenyl product.

The methods described herein may also be used for preparation ofisotopically labeled compounds, by employing ¹⁴C-containing carbonnucleophiles.

III. Therapeutic Compositions

Formulations containing the triptolide derivatives of the invention maytake the form of solid, semi-solid, lyophilized powder, or liquid dosageforms, such as tablets, capsules, powders, sustained-releaseformulations, solutions, suspensions, emulsions, ointments, lotions, oraerosols, preferably in unit dosage forms suitable for simpleadministration of precise dosages. The compositions typically include aconventional pharmaceutical carrier or excipient and may additionallyinclude other medicinal agents, carriers, or adjuvants. Preferably, thecomposition will be about 0.5% to 75% by weight of a compound orcompounds of the invention, with the remainder consisting of suitablepharmaceutical excipients. For oral administration, such excipientsinclude pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharine, talcum, cellulose, glucose, gelatin,sucrose, magnesium carbonate, and the like. If desired, the compositionmay also contain minor amounts of non-toxic auxiliary substances such aswetting agents, emulsifying agents, or buffers.

The composition may be administered to a subject orally, transdermallyor parenterally, e.g., by intravenous, subcutaneous, intraperitoneal, orintramuscular injection. For use in oral liquid preparation, thecomposition may be prepared as a solution, suspension, emulsion, orsyrup, being supplied either in liquid form or a dried form suitable forhydration in water or normal saline. For parenteral administration, aninjectable composition for parenteral administration will typicallycontain the triptolide derivative in a suitable intravenous solution,such as sterile physiological salt solution.

Liquid compositions can be prepared by dissolving or dispersing thetriptolide derivative (about 0.5% to about 20%) and optionalpharmaceutical adjuvants in a pharmaceutically acceptable carrier, suchas, for example, aqueous saline, aqueous dextrose, glycerol, or ethanol,to form a solution or suspension. The high water solubility of thecompounds of the invention make them particularly advantageous foradministering in aqueous solution, e.g. by intraperitoneal injection.Although aqueous solutions are preferred, compositions in accordancewith the invention may also be formulated as a suspension in a lipid(e.g., a triglyceride, a phospholipid, or a polyethoxylated castor oilsuch as “CREMOPHOR EL”), in a liposomal suspension, or in an aqueousemulsion.

The compound may also be administered by inhalation, in the form ofaerosol particles, either solid or liquid, preferably of respirablesize. Such particles are sufficiently small to pass through the mouthand larynx upon inhalation and into the bronchi and alveoli of thelungs. In general, particles ranging from about 1 to 10 microns in size,and preferably less than about 5 microns in size, are respirable. Liquidcompositions for inhalation comprise the active agent dispersed in anaqueous carrier, such as sterile pyrogen free saline solution or sterilepyrogen free water. If desired, the composition may be mixed with apropellant to assist in spraying the composition and forming an aerosol.

Methods for preparing such dosage forms are known or will be apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (19th Ed., Williams & Wilkins, 1995). The composition to beadministered will contain a quantity of the selected compound in aneffective amount for effecting immunosuppression in a subject orapoptosis in a targeted cell.

As described, for example, in Panchagnula et al. (2000), the partitioncoefficient or logP of a pharmaceutical agent can affect its suitabilityfor various routes of administration, including oral bioavailability.The compounds described herein have higher calculated logP values thanthe parent compound, triptolide (see Table), making them bettercandidates for oral availability. (Compounds represented in the Tableare compounds of formula I where R² is OH, CR³R⁵ and CR⁴R⁶ are both CH₂,and R¹ is as indicated. As described above, the stereochemistry at C14in these compounds is such that the hydroxyl group is depicted above theplane of the page.) Compounds LogP ^(a) LogP ^(b) Triptolide (R¹ = H)−0.08 0.27 R¹ = —CH₃ (PG670) 0.14 0.34 R¹ = —CH═C═CH₂ 0.63 0.89 R¹ =—CH₂CH═CH 0.77 0.99 R¹ = —CH₂—C≡CH 0.30 0.53 R¹ = —C≡CH 0.02 0.28 R¹ =—CH═CH₂ 0.49 0.74 R¹ = —(CH₂)₅CH₃ 2.30 2.40^(a) calculated per A. K. Ghose et al., J. Chem. Inf. Comput. Sci. 27:21-35(1987); SD = 0.47^(b) calculated per V. N. Viswanadhan, J. Chem. Inf. Comput. Sci. 29:163 (1989); SD = 0.49IV. Immunomodulating and Antiinflammatory Treatment

Immunoregulatory abnormalities have been shown to exist in a widevariety of autoimmune and chronic inflammatory diseases, includingsystemic lupus erythematosis, chronic rheumatoid arthritis, type I andII diabetes mellitus, inflammatory bowel disease, biliary cirrhosis,uveitis, multiple sclerosis and other disorders such as Crohn's disease,ulcerative colitis, bullous pemphigoid, sarcoidosis, psoriasis,ichthyosis, Graves ophthalmopathy and asthma. Although the underlyingpathogenesis of each of these conditions may be quite different, theyhave in common the appearance of a variety of autoantibodies andself-reactive lymphocytes. Such self-reactivity may be due, in part, toa loss of the homeostatic controls under which the normal immune systemoperates.

Similarly, following a bone-marrow or an organ transplantation, the hostlymphocytes recognize the foreign tissue antigens and begin to produceantibodies which lead to graft rejection.

One end result of an autoimmune or a rejection process is tissuedestruction caused by inflammatory cells and the mediators they release.Anti-inflammatory agents such as NSAID's act principally by blocking theeffect or secretion of these mediators but do nothing to modify theimmunologic basis of the disease. On the other hand, cytotoxic agents,such as cyclophosphamide, act in such a nonspecific fashion that boththe normal and autoimmune responses are shut off. Indeed, patientstreated with such nonspecific immunosuppressive agents are as likely tosuccumb from infection as they are from their autoimmune disease.

The compositions of the present invention are useful in applications forwhich triptolide and its prodrugs and other derivatives have proveneffective, e.g. in immunosuppression therapy, as in treating anautoimmune disease, preventing transplantation rejection, or treating orpreventing graft-versus-host disease (GVHD). See, for example, co-ownedU.S. Pat. No. 6,150,539, which is incorporated herein by reference.Triptolide and the present derivatives are also useful for treatment ofother inflammatory conditions, such as traumatic inflammation, and inreducing male fertility.

The method is useful for inhibiting rejection of a solid organtransplant, tissue graft, or cellular transplant from an incompatiblehuman donor, thus prolonging survival and function of the transplant,and survival of the recipient. This use would include, but not belimited to, solid organ transplants (such as heart, kidney and liver),tissue grafts (such as skin, intestine, pancreas, gonad, bone, andcartilage), and cellular transplants (such as cells from pancreas, brainand nervous tissue, muscle, skin, bone, cartilage and liver).

The method is also useful for inhibiting xenograft (interspecies)rejection; i.e. in preventing the rejection of a solid organ transplant,tissue graft, or cellular transplant from a non-human animal, whethernatural in constitution or bioengineered (genetically manipulated) toexpress human genes, RNA, proteins, peptides or other non-native,xenogeneic molecules, or bioengineered to lack expression of theanimal's natural genes, RNA, proteins, peptides or other normallyexpressed molecules. The invention also includes the use of acomposition as described above to prolong the survival of such a solidorgan transplant, tissue graft, or cellular transplant from a non-humananimal.

In another aspect, the invention includes a method of treatment orprevention of graft-versus-host disease, resulting from transplantationinto a recipient of matched or mismatched bone marrow, spleen cells,fetal tissue, cord blood, or mobilized or otherwise harvested stemcells. The dose is preferably in the range 0.25-2 mg/kg body weight/day,preferably 0.5-1 mg/kg/day, given orally or parenterally.

Also included are methods of treatment of autoimmune diseases ordiseases having autoimmune manifestations, such as Addison's disease,autoimmune hemolytic anemia, autoimmune thyroiditis, Crohn's disease,diabetes (Type I), Graves' disease, Guillain-Barre syndrome, systemiclupus erythematosus (SLE), lupus nephritis, multiple sclerosis,myasthenia gravis, psoriasis, primary biliary cirrhosis, rheumatoidarthritis and uveitis, asthma, atherosclerosis, Type I diabetes,psoriasis, and various allergies. In treating an autoimmune condition,the patient is given the composition on a periodic basis, e.g., 1-2times per week, at a dosage level sufficient to reduce symptoms andimprove patient comfort. For treating rheumatoid arthritis, inparticular, the composition may be administered by intravenous injectionor by direct injection into the affected joint. The patient may betreated at repeated intervals of at least 24 hours, over a several weekperiod following the onset of symptoms of the disease in the patient.

Immunosuppressive activity of compounds in vivo can be evaluated by theuse of established animal models known in the art. Such assays may beused to evaluate the relative effectiveness of immunosuppressivecompounds and to estimate appropriate dosages for immunosuppressivetreatment. These assays include, for example, a well-characterized ratmodel system for allografts, described by Ono and Lindsey (1969), inwhich a transplanted heart is attached to the abdominal great vessels ofan allogeneic recipient animal, and the viability of the transplantedheart is gauged by the heart's ability to beat in the recipient animal.A xenograft model, in which the recipient animals are of a differentspecies, is described by Wang (1991) and Murase (1993). A model forevaluating effectiveness against GVHD involves injection of normal F₁mice with parental spleen cells; the mice develop a GVHD syndromecharacterized by splenomegaly and immunosuppression (Korngold, 1978;Gleichmann, 1984). Single cell suspensions are prepared from individualspleens, and microwell cultures are established in the presence andabsence of concanavalin A to assess the extent of mitogenicresponsiveness.

For therapy in transplantation rejection, the method is intendedparticularly for the treatment of rejection of heart, kidney, liver,cellular, and bone marrow transplants, and may also be used in thetreatment of GVHD. The treatment is typically initiated perioperatively,either soon before or soon after the surgical transplantation procedure,and is continued on a daily dosing regimen, for a period of at leastseveral weeks, for treatment of acute transplantation rejection . . .During the treatment period, the patient may be tested periodically forimmunosuppression level, e.g., by a mixed lymphocyte reaction involvingallogenic lymphocytes, or by taking a biopsy of the transplanted tissue.

In addition, the composition may be administered chronically to preventgraft rejection, or in treating acute episodes of late graft rejection.As above, the dose administered is preferably 1-25 mg/kg patient bodyweight per day, with lower amounts being preferred for parenteraladministration, and higher amounts for oral administration. The dose maybe increased or decreased appropriately, depending on the response ofthe patient, and over the period of treatment, the ability of thepatient to resist infection.

Also within the scope of the invention is a combination therapycomprising a compound of formula I and one or more conventionalimmunosuppressive agents. These immunosuppressant agents within thescope of this invention include, but are not limited to, IMUREK™(azathioprine sodium), brequinar sodium, SPANIDIN™ (gusperimustrihydrochloride, also known as deoxyspergualin), mizoribine (also knownas bredinin), CELLCEPT™ (mycophenolate mofetil), NEORAL™ (Cyclosporin A;also marketed as a different formulation under the trademarkSANDIMMUNE™), PROGRAF™ (tacrolimus, also known as FK-506), RAPIMMUNE™(sirolimus, also known as rapamycin), leflunomide (also known asHWA-486), ZENAPAX™, glucocortcoids, such as prednisolone and itsderivatives, antibodies such as orthoclone (OKT3), and antithymyocyteglobulins, such as thymoglobulins. The compounds are useful aspotentiators when administered concurrently with anotherimmunosuppressive drug for immunosuppressive treatments as discussedabove. A conventional immunosuppressant drug, such as those above, maythus be administered in an amount substantially less (e.g. 20% to 50% ofthe standard dose) than when the compound is administered alone.Alternatively, the triptolide derivative and immunosuppresive drug areadministered in amounts such that the resultant immunosuppression isgreater than what would be expected or obtained from the sum of theeffects obtained with the drug and triptolide derivative used alone.Typically, the immunosuppressive drug and potentiator are administeredat regular intervals over a time period of at least 2 weeks.

The compositions and method of the invention are also useful for thetreatment of inflammatory conditions such as asthma, both intrinsic andextrinsic manifestations. For treatment of asthma, the composition ispreferably administered via inhalation, but any conventional route ofadministration may be useful. The composition and method may also beused for treatment of other inflammatory conditions, including traumaticinflammation, inflammation in Lyme disease, psoriasis, chronicbronchitis (chronic infective lung disease), chronic sinusitis, sepsisassociated acute respiratory distress syndrome, Behcet's disease,pulmonary sarcoidosis, pemphigus, pemphigoid inflammatory bowel disease,and ulcerative colitis.

The compositions of the invention may also be administered incombination with a conventional anti-inflammatory drug (or drugs), wherethe drug or amount of drug administered is, by itself, ineffective toinduce the appropriate suppression or inhibition of inflammation.

The dose that is administered is preferably in the range of 1-25 mg/kgpatient body weight per day, with lower amounts being preferred forparenteral administration, and higher amounts being preferred for oraladministration. Optimum dosages can be determined by routineexperimentation according to methods known in the art.

V. Anticancer Treatment

Triptolide derivatives have shown effectiveness in cancer treatment.See, for example, co-owned U.S. Pat. No. 6,620,843, incorporated hereinby reference, which describes high efficacy of a triptolide derivative,in comparison to 5-FU and CPT-11, in inhibiting tumor growth in studieswith tumor xenografts of the HT-29 human colon cancer cell line. Thetriptolide derivative (triptolide 14-succinate) strongly inhibited tumorgrowth, to a significantly greater degree than 5-FU and CPT-11, andinduced tumor regression.

The invention thus includes the use of compositions as described aboveto treat cancers, including cancers involving cells derived fromreproductive tissue (such as Sertoli cells, germ cells, developing ormore mature spermatogonia, spermatids or spermatocytes and nurse cells,germ cells and other cells of the ovary), the lymphoid or immune systems(such as Hodgkin's disease and non-Hodgkin's lymphomas), thehematopoietic system, and epithelium (such as skin and gastrointestinaltract), solid organs, the nervous system, and musculo-skeletal tissue.The triptolide derivatives may be used for treatment of various cancercell types, including, but not limited to, breast, colon, small celllung, large cell lung, prostate, malignant melanoma, liver, kidney,pancreatic, esophogeal, stomach, ovarian, cervical or lymphoma tumors.Treatment of breast, colon, lung, and prostate tumors is particularlycontemplated. Treatment of leukemias is also contemplated. Thecomposition may be administered to a patient afflicted with cancerand/or leukemia by any conventional route of administration, asdiscussed above.

The method is useful to slow the growth of tumors, prevent tumor growth,induce partial regression of tumors, and induce complete regression oftumors, to the point of complete disappearance. The method is alsouseful in preventing the outgrowth of metastases derived from solidtumors.

The compositions of the invention may be administered as sole therapy orwith other supportive or therapeutic treatments not designed to haveanti-cancer effects in the subject. The method also includesadministering the invention compositions in combination with one or moreconventional anti-cancer drugs or biologic protein agents, where theamount of drug(s) or agent(s) is, by itself, ineffective to induce theappropriate suppression of cancer growth, in an amount effective to havethe desired anti-cancer effects in the subject. Such anti-cancer drugsinclude actinomycin D, camptothecin, carboplatin, cisplatin,cyclophosphamide, cytosine arabinoside, daunorubicin, doxorubicin,etoposide, fludarabine, 5-fluorouracil, hydroxyurea, gemcitabine,irinotecan, methotrexate, mitomycin C, mitoxantrone, paclitaxel,taxotere, teniposide, topotecan, vinblastine, vincristine, vindesine,and vinorelbine. Anti-cancer biologic protein agents include tumornecrosis factor (TNF), TNF-related apoptosis inducing ligand (TRAIL),other TNF-related or TRAIL-related ligands and factors, interferon,interleukin-2, other interleukins, other cytokines, chemokines, andfactors, antibodies to tumor-related molecules or receptors (such asanti-HER2 antibody), and agents that react with or bind to these agents(such as members of the TNF super family of receptors, other receptors,receptor antagonists, and antibodies with specificity for these agents).

EXAMPLES

The following examples are intended to illustrate but not in any waylimit the invention.

Example 1 Preparation of 14-C-methyltriptolide (PG670)

To a solution of triptonide, PG492 (60 mg, 0.17 mmol) in THF (5 ml) at−78° C. was added 0.45 ml of methyl lithium (1.4 M solution in ethylether, 0.63 mmol, 3.7 eq) under N₂. The solution was stirred at −78° C.for 2 hrs. 45 mins. and then at room temperature for 2 hrs., at whichtime the starting material had disappeared on TLC. Acetic acid (1 ml)was slowly added. The solution was then concentrated under vacuum. Thecrude product was dissolved in dichloromethane (3 ml) and passed througha pad of silica gel, which was then washed with 5% methanol in ethylacetate (80 ml). After removal of solvent, 78 mg of crude product wasobtained. This was dissolved in acetonitrile (0.6 ml) and filtered. Theproduct mixture was separated on HPLC, using a 10×250 mm column ofEconosil C18 (5μ) and a guard column cartridge (7.5×4.6 mm) of AlltimaC18 (5μ), both from Alltech, with mobile phase CH₃CN/H₂O 40/60 with aflow rate of 2.0 ml/min. The sixth peak, having a retention time of32.13 mins., was collected and concentrated under vacuum. The producthad m/z 374 (7.9 mg, yield: 12.6%).

The structure was verified by NMR. H¹ NMR (300 MHz, CDCl₃): δ=4.68 (2H,s, 19-CH₂), 3.95 (1H, d, 11-CH), 3.50 (1H, d, 12-CH), 3.49 (1H, s,14-OH), 3.39 (1H, d, 7-CH), 2.69 (1H, m, 5-CH), 2.46 (1H, m, 15-CH),2.33 (1H, m, 2-CHb), 2.12 (1H, m, 6-CHb), 1.97 (1H, m, 6-CHa), 1.57 (1H,dd, 1-CHb), 1.38 (1H, m, 2-CHa), 1.22 (1H, m, 1-CHa), 1.14 (3H, s,14-CH₃ or 20-CH₃), 1.09 (3H, d, 17-CH₃), 1.08 (3H, s, 20-CH₃ or 14-CH₃),0.81 (3H, d, 16-CH₃) ppm.

Example 2 Apoptosis Assays

A. Incubation of Compounds with Human Serum

Pooled human serum was stored in aliquots at −80° C. Test compounds wereadded at 20 mM to thawed human serum in 1.5 ml microfuge tubes andincubated at 37° C. in a water bath for 48 hours. The test samples wereplaced on ice until dilution for the bioassay. Controls consisted of thecompounds incubated in complete tissue culture medium (RPMI 1640 mediumplus 5% heat-inactivated fetal calf serum, 1% HEPES, 1% pen/strep, 1%glutamine) rather than human serum.

B. Annexin V Apoptosis Assay.

Test samples (incubated in human serum as described in section A) werediluted to 1 mM in complete tissue culture medium. Aliquots were placedin microculture plates and serial dilutions were prepared so that thefinal concentration would encompass the range of 2 to 6,000 nM withhalf-log increments. Cells from an exponentially expanding culture ofthe Jurkat human T lymphocyte cell line (#TIB-152 obtained from AmericanType Culture Collection, Manassas, Va.) were harvested, washed once bycentrifugation and dilution in complete tissue culture medium, anddiluted to a concentration of 1×10⁶ cells/ml. A volume of 100 μl ofJurkat cells (1×10⁵ cells) was added to wells containing 100 μl of thediluted compounds, and the plates were incubated at 37° C. in a 5% CO₂incubator.

After 24 hours, the plates were centrifuged to pellet the cells, and thecells were washed twice with 2% heat-inactivated fetal calf serum inPBS. To each well, 500 ul of binding buffer was added, according to theAnnexin V assay procedure (BioVision, Inc., Mountain View, Calif.).Next, 5 μl of the fluorescein isothiocyanate (FITC) conjugate of AnnexinV (BioVision, Inc.) was added to each well, followed by 5 minutes ofincubation in the dark. In some assays, propidium iodide (BioVision,Inc.) was added at this stage to check for necrotic cells. The contentsof the wells were individually transferred into test tubes, andapoptosis was analyzed using a FACSCalibur flow cytometer (BDImmunocytometry Systems, San Jose, Calif.). Cells positive for Annexin Vbinding were considered to be apoptotic, and the data were calculated aspercent apoptotic cells.

Data were plotted as the concentration of compound incubated in serumversus percent apoptotic cells. The results for PG670 (14-methyltriptolide), compared with PG490 (triptolide) and solvent controls, aregiven in FIG. 1.

C. Terminal Deoxynucleotidyl Transferase Apoptosis Assay

In this assay, test samples are prepared as described in section A aboveand diluted as described in section B. Jurkat human T lymphocyte cellsare added, also as described in section B, and the plates are incubatedat 37° C. in a 5% CO₂ incubator. After 24 hours, the plates arecentrifuged to pellet the cells, and the cells are washed with PBS. Thecells are fixed in paraformaldehyde, washed, treated with ethanol,washed and then incubated with the enzyme terminal deoxynucleotidyltransferase (TdT) and fluorescein labeled-deoxyuridine (dUTP). Theprocess allows 3′ end labeling of DNA molecules nicked during the DNAfragmentation phase of apoptosis (terminal deoxynucleotidyl transferasedUTP nick end labeling; TUNEL labeling). The cells are washed, treatedwith ribonuclease, washed and resuspended in medium containing propidiumiodide to distinguish intact apoptotic cells. The contents of the assaywells are individually transferred into test tubes, and apoptosis levelsare analyzed using a FACSCalibur flow cytometer (BD ImmunocytometrySystems, San Jose, Calif.). Cells positive for Fl-dUTP are considered tobe apoptotic.

Example 3 IL-2 Production Assay

Test samples, incubated in human serum as described in Example 2A, werediluted to 1 mM in complete tissue culture medium. Aliquots were placedin microculture plates that had been coated with anti-CD3 antibody (usedto stimulate the production of IL-2 by Jurkat cells), and serialdilutions were prepared so that the final concentration would encompassthe range of 0.001 to 10,000 nM in log increments. Cells from anexponentially expanding culture of the Jurkat human T lymphocyte cellline (#TIB-152 obtained from American Type Culture Collection, Manassas,Va.) were harvested, washed once by centrifugation and dilution incomplete tissue culture medium, and diluted to a concentration of 2×10⁶cells/ml. A volume of 50 μl of Jurkat cells (1×10⁵ cells) was added towells containing 100 μl of the diluted compounds, 50 μl of PMA (10ng/ml) was added to each well, and the plates were incubated at 37° C.in a 5% CO₂ incubator. After 24 hours, the plates were centrifuged topellet the cells, 150 μl of supernatant was removed from each well, andthe samples were stored at −20° C. The stored supernatants were analyzedfor human IL-2 concentration using the Luminex 100 (Luminex Corporation,Austin, Tex.), Luminex microspheres coupled with anti-IL-2 captureantibody, and fluorochrome-coupled anti-IL-2 detection antibody. Thedata were expressed as ng/ml of IL-2.

The data were plotted as the concentration of compound incubated inserum versus IL-2 concentration. The results for PG670 (14-methyltriptolide), compared with PG490 (triptolide) and a solvent control, aregiven in FIG. 2.

1-8. (canceled)
 9. A method of effecting immunosuppression, comprisingadministering to a subject in need of such treatment, in apharmaceutically acceptable vehicle, an effective amount of a compoundhaving the structure I:

where: R¹ is H or R, where R is selected from lower alkyl, alkenyl,alkynyl, and allenyl, or R¹ together with R²=O (oxo); R²=OH or R¹ and R²together=O (oxo); CR³R⁵ and CR⁴R⁶ are selected from CH₂, CHOH and CROH;at least one of R¹, R⁵ and R⁶ is R; and at least one of CR³R⁵ and CR⁴R⁶is CH₂.
 10. A method of inducing apoptosis in a cell, comprisingcontacting said cell with an effective amount of a compound having thestructure I:

where: R¹ is H or R, where R is selected from lower alkyl, alkenyl,alkynyl, and allenyl, or R¹ together with R²=O (oxo); R²=OH or R¹ and R²together=O (oxo); CR³R⁵ and CR⁴R⁶ are selected from CH₂, CHOH and CROH;at least one of R¹, R⁵ and R⁶ is R; and at least one of CR³R⁵ and CR⁴R⁶is CH₂.